The insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IFG-II/CI-MPR) is a multifunctional protein that binds both IGF-II and lysosomal enzymes and, along with the cation-dependent MPR (CD-MPR), mediates the targeting of newly synthesized lysosomal enzymes to the lysosome. Our recent studies have demonstrated the polarized expression of the MPRs in the enterocyte-like cell line, Caco-2. In addition, we have found that the IGF-II/CI-MPR expressed on the apical surface of Caco-2 cells, unlike the receptor expressed on the basolateral surface, is unable to endocytose lysosomal enzymes. The objectives of the proposal are to determine: 1) the factors which regulate the intra cellular sorting of the MPRs in polarized epithelial cells, and 2) the molecular/cellular basis for the failure of the IGF-II/CI-MPR to internalize ligands from the apical surface of enterocytes. To achieve these goals, pulse-chase labeling studies on filter grown Caco-2 cells in conjunction with domain-specific cell surface labeling techniques will be performed to determine the kinetics and intracellular pathways of targeting MPRs to the apical and basolateral surfaces. The region(s) of the MPRs' cytoplasmic domain important for their intracellular targeting will be identified by transfecting mutant MPR cDNAs into Caco-2 cells and analyzing the mutant MPRs' sorting and steady state surface distribution. To probe the molecular/cellular basis for the expression of functionally distinct IGF-II/CI-MPRs on the apical and basolateral surface, the receptor from isolated apical and basolateral membranes will be characterized with respect to size, presence of an intact cytoplasmic domain, presence and type of N-linked oligosaccharides, susceptibility to proteolysis, and the ability to bind and internalize ligands in different membrane environments. Taken together, these studies will lead to a better understanding of the functional expression of the MPRs that are essential for the biogenesis of lysosomes, an organelle important for regulating protein turnover in absorptive enterocytes. These studies will also address our long-term goals of identifying: 1) the factors involved in the establishment and maintenance of polarity in enterocytes, and 2) the role of IGF-II.CI-MPR in enterocyte differentiation and proliferation.